AbstractFish‐scale iridophore cells deposit guanine crystals and assemble them into multilayer reflectors to produce silvery reflectance. The crystal orientation controls the reflective properties of the fish scales, but little is known about the degree of orientation of the guanine crystals and whether this orientation is pre‐determined at the level of an individual cell. Koi fish‐scale‐attached iridophores, iridophores on regenerated scales, and cultured iridophores were examined by using light microscopy and synchrotron micro‐X‐ray diffraction. More than 95 % of the thin {100} guanine crystal plates in the iridophores of the mature and regenerated scales are oriented parallel to the scale surface and perpendicular to the direction of the incoming light. More than 70 % of the crystals in cultured iridophore cells are also in this orientation. The crystals are elongated and within each cell on the mature scale and in the cultured cells the long morphological axes are well aligned with the long axis of the iridophore. In contrast to the cultured iridophores, in the mature scale the iridophore cells are co‐aligned with each other. Cultured iridophores are flexible and motile, and azimuthal crystal orientations vary as the cells move. We conclude that iridophore cells function as independent units and that the control over crystal orientation is pre‐determined at the individual cell level in the direction that is essential for function, namely, exposing the large reflecting crystal surface to light.